Project Summary/Abstract Donor-specific transplantation tolerance, as defined by graft acceptance after discontinuation of immunosuppressive therapies, can be achieved in specific pathogen-free rodents but rarely in humans. Deletion, anergy and suppression of alloreactive T cells are 3 cellular mechanisms most often associated with the induction of transplantation tolerance in mice. It has been shown that deletion of alloreactive T cells and suppression of remaining alloreactive T cells by CD25+ regulatory T cells (Tregs) are necessary for the induction of transplantation tolerance by costimulation blockade. However, whether T cell anergy is necessary and/or sufficient for the induction and/or the maintenance of transplantation tolerance is not known. Furthermore, it is not clear if these 3 mechanisms of T cell tolerance (and others potentially) need to be coinduced simultaneously for tolerance to be robust and maintained long-term successfully. If they do need to be co-induced, the usual lack of development of transplantation tolerance in humans could be due to the inability of current immunosuppressive regimens to induce/maintain these 3 pathways concurrently. Our preliminary experiments indicate that tolerance is not an all or none phenomenon and that a readout of allograft acceptance is not a sensitive measure of the functional robustness of transplantation tolerance. Indeed, we observed that depletion of Tregs or blockade of PD-L1 does not abrogate established tolerance, but is able to precipitate acute cardiac allograft rejection in tolerant mice that had been previously infected with Listeria monocytogenes but retained their graft. This suggests that the quality or quantity of mechanisms that maintain graft acceptance are modified after infection and that a simple lack of acute rejection does not inform on the stability of the tolerant state or how close the alloimmune response is to the rejection threshold. We propose that transplantation tolerance is a continuum of states in which the number and intensity of anti-inflammatory processes that constrain alloimmunity are progressively eroded by pro-inflammatory signals (infectious episodes or others) until a rejection threshold is reached. Although intuitive, this concept has not been investigated in any systematic manner and has important implications in clinical transplantation: it may explain why patients who achieve transplantation tolerance, i.e., years of stable function without immunosuppressive drugs, or even patients stably maintained with minimal immunosuppression, may suddenly reject their grafts; the robustness of the tolerance level may also determine whether immunosuppressive drugs can be withdrawn and whether tolerance can persist. We specifically hypothesize that a state of transplantation tolerance that is robust and that can persist and resist Inflammatory insults may only be achieved when T cell depletion, T cell regulation and T cell anergy are all co-induced simultaneously and optimally. Insights gained from these mechanistic analyses will be of importance in the clinic, where the genetic diversity of the transplant population all but guarantees that individuals receiving the same treatment will develop different degrees of deletion, anergy and suppression. This hypothesis will be tested in the context of the following specific aims: Aim 1: To determine the requirement for T cell anergy in the induction and maintenance of transplantation tolerance. Aim 2: To assess the necessity and sufficiency of T cell mechanisms of tolerance in the establishment of robust transplantation tolerance.